Recombination of uracil-containing lambda bacteriophages.

Abstract

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.